The Immune Study Of Fusion Protein APB-DOCK8 Transgenic Tomato Anti-caries Vaccine In SD Rats

Objective: Based on the plasmid p ET30a-PAc A and the T₀ seeds contained exogenous gene APB-DOCK8 transgenic tomato,to prepare polyclonal antibody,identify the transgenic tomato plant and its fruit and detect fusion protein using techniques of PCR,Western and El ISA.This experiment detects the specific antibody of animals induced by oral immunization with fusion protein of the transgenic plants.Method: The study contains three experiments.Experiment 1: Expression of the recombinant vector-pET30a-PAcA,protein purification and antibody preparation.1.A transformant E.Coli BL21?DE3?containing the recombiant plasmid pET30a-PAcA was obtained,IPTG was added to induce protein expression.2.Decompose the induced E.Coli BL21 to obtain the target protein,purify the protein by Ni-NTA spin columns,then identify the purified protein.3.After the immunization of New Zealand white rabbits with PAcA,collect blood,separate serum and obtain the polyclonal antibody of target protein.Experiment 2: Gene identification and fusion protein detection of APB-DOCK8 transgenic tomato fruit.1.This experiment explored how to obtain the high quality DNA of tomato fruit,we compared the effects of improved CTAB method and DNA extraction kit.Plants containing exogenous gene APB-DOCK8 were identified by detecting the GUS gene of transgenic tomato.2.The total protein of the identified transgenic T₀&T₁ tomatoes were quantitated by BCA method,the fusion protein concentration was detected by ELISA,the expression of the protein was analyzed by Western blotting.Compare the express level of the fusion protein with E8 trangenetic tomatoes without specific promoter.Experiment 3: Oral immunization with fusion protein APB-DOCK8 of transgenic tomato in SD rats.1.Animal selection and grouping: 18-year-old SD female rats?n=24?,randomly divided into three groups: positive control group?n=8?,experimental group?n=8?and negative control group?n=8?.Dental caries model was established.2.Immune methods,antigen and dose: the experimental group fed with 10 ml / kg dose of transgenic tomato juice which containing fusion protein?containing fusion protein365.52 ?g?for immunization,the negative control group was fed with normal group of non-transgenic tomato juice at 10 m L/kg dose,while the positive control group was fed with S.mutans UA159 inactivated vaccine immunization?1 m L each time,the concentra--tion of bacteria was 1×109CFU/m L?.3.Immune times: Immunize SD rats once a week since the 24 th day,total for 4 weeks.4.Sample collection: Collected saliva and blood samples of SD rats before the first immunization and on the 7th day after each immunization?until the 12 th week?,samples will not be collected in week 9 and week 11.5.Detection of specific antibodies: indirect ELISA method was conducted to detect SIg A in saliva and Ig G in serum.6.Sacrificed the animal then took its important organs for histopathological examination.Caries score according to the classic Keyes scoring method by its maxilla and mandible.Results:1.Optimized the conditions of prokaryotic expression,obtained purified BL21/p ET30a-PAc A protein and detected its concentrations,the protein concentration was0.5616 mg/m L,prepared the polyclonal antibody of the protein.2.Improved CTAB method is more efficient than the kit method when made high-throughput DNA extractions from transgenic tomatoes.GUS gene was used to detect transgenic tomatoes,15 out of 20 transgenic tomato plants showed specific bands,accounting for 75% of the total plants.3.The total protein concentrations of the transgenic T₀&T₁ tomato fruit was 13.57mg/m L & 13.43 mg/m L,respectively.The fusion protein concentrations of the transgenic T₀&T₁ tomato fruit was 41.372 ?g/m L,36.552 ?g/m L,respectively.The proportion of exogenous protein in the transgenic T₀&T₁ tomato fruit was 0.3048%,0.2722%,respectively.Western blotting showed that the transgenic tomato protein samples showed a specific band of 85 KD,which coincided with the expected size of APB fusion protein.4.After 7 days of immunization,immune responses was detected in each group.In the experimental group,the level of Ig G in blood serum and SIg A in saliva increased gradually;the level of antibody reached its peak in week 6,then became stable during week 7&8 and declined significantly during week 8.From week 0 to week 12,the specific SIg A antibody was not significant between the experimental group and the positive control group?P>0.05?.During week 10,the specific Ig G was statistically significant between the experimental group and the positive control group?P<0.05?,the antibody level on the other time points were not significant;From week 1 to week 10,the specific antibody level in positive control group and experimental control group was statistically significant when compared to the negative control group.Comparisons of caries lesion ware made between the positive control group and the experimental group,there was no statistical difference except the Ds level?P>0.05?,there was statistical difference of dental caries scorning between the positive control group and the experimental group(?P<0.05?.5.General safety testing: There was no significant differences in the body weight of SD rats between different group at the same time?P>0.05?.No significant pathological changes were observed in the main organs of animals in the positive control group,the experimental group and the negative control group.Conclusion:1.The recombinant plasmid p ET30a-PAc A successfully expressed the soluble BL21/p ET30a-PAc A protein.The anti-PAc A polyclonal antibody was successfully prepared,providing a solid foundation for detecting the target protein in transgenic tomato.2.Positive plants expressing exogenous APB-DOCK8 were screened out from the T₁ transgenic tomato plants.Although the expression of APB-DOCK8 appeared fractured,both T₀&T₁ transgenic tomato plants were able to express APB protein,and the positive expression rate and expression efficiency were high.The expression of APB protein was stable.3.APB-DOCK8 transgenic tomato has immunogenicity.Immunization of SD rats with transgenic tomato juice containing recombinant protein triggered mucosal immunity and systemic immune response,producing high levels of specific antibodies in saliva and blood,and inhibits the occurrence of dental caries.At the same time,animals did not show obviously physical harm after immunization.